Controlled regeneration of zea diploperennis hybrids and breeding strategies therewith

ABSTRACT

This invention provides a method for high frequency plant regeneration from somatic stem donor tissue of field grown Zea diploperennis, a diploid, perennial corn ancestor with high tillering capacity. This species is used as a parent in a maize improvement strategy to transfer the unique traits of high tillering and plantlet regeneration capacity into cultivated corn. After 3-4 subcultures of cultured somatic tissues on a primary medium, small callus fragments are transferred to a secondary medium devoid of the auxin, 2,4-D. After a few days, numerous shoots regenerate and develop into normal plantlets which are then separated and transferred to a tertiary medium for root development. The selection of somaclonal variants form cultured somatic cells of interspecific hybrids between corn and teosinte are used for the synthesis of unique breeding lines suited for development of improved corn varieties. A protocol for gene transfer employing recombinant DNA techniques is also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.597,472 filed Apr. 6, 1984 now U.S. Pat. No. 4,659,668.

FIELD OF THE INVENTION

This invention relates to the field of improvement of agricultural cropspecies. More specifically, the invention provides a method for theregeneration of an ancesteral corn species and for the use of theregenerated plants in a commercial corn improvement program.

BACKGROUND OF THE INVENTION

The perfection of plant regeneration capabilities and the application ofbiotechnological techniques for genome modification provide a highlydesirable system for the improvement of crop species.

Investigations concerning the morphogenesis of plant tissue in culturedate back at least to the 1950's (Skoog, F. and Miller C. O., Symp Soc.Exp. Biol., 11:118 (1957)) and have continued apace to date. Severalmonographs provide extensive reviews of the field and containcompilations of numbers of species which will undergo plant regenerationin culture (See for example, Murashige T., In: "Propagation of HigherPlants through Tissue Culture," T. A. Thorpe, Ed., p. 15, Univ. CalgaryPress, Calgary (1978); Vasil, I. K. et al. Adv. Gent. 20:127 (1979) andEvans, D. A., et al. In: "Plant Tissue Culture: Methods and Applicationsin Agriculture: T. A. Thorpe, Ed. pg. 45, Academic Press, N.Y. (1981)).

The impressive list of plants species cited in the above-referencedmonographs, for which successful regeneration has been achieved, beliesthe difficulties in achieving those results. As will be noted later,successful regeneration of a particular species is often characterizedby the addition of (or even omission of) catalytic amounts of auxins,cytokinins, or other growth regulators. Further, successful regenerationmay also be a function of not only the mere presence of a certaincompound but its ratio to other media components as well. Since eachplant species appears to possess a relatively unique optimal set ofmedia requirements, the successful preparation and regeneration of a newspecies cannot be necessarily inferred from the successful regimensapplied to unrelated plant varieties.

Despite the recent advances in plant regeneration for a variety ofspecies, corn (Zea mays) is one of the crops which has been refractoryto regeneration protocols; hence, the application of plant cell culturefor improvement of the majority of pure lines and commercial hybrids ofthis crop has lagged behind that progress in the field in general.

Absent a functioning regeneration protocol, more traditional avenues forcrop improvement have been utilized. One approach has been to introduceinto the commercial corn genome agronomically useful characteristicsderived from exotic or "wild" Zea germplasm by conventional sexualhybridization and back-crossing breeding procedures. One source ofexotic germplasm which has been employed is teosinte (Zea spp.) Teosintehas been described as a possible ancestor of today's modern Zea maysforms. There are several known races of teosinte that are either annualand diploid or perennial and tetraploid. A new teosinte that is bothdiploid and perennial (Zea diploperennis) has recently been discovered(Iltis, H. H., et al. Science 203: 186-188 (1979)). This species can besexually hybridized with commercial corn. Interspecies hybrids betweenZ. mays and Z. diploperennis are fertile and based on nearly completechromosome pairing will be potentially useful for crop improvement(Pasupulet, C. V. and W. C. Galinat, J. of Heredity 73:168-70 (1982) andGalinat, W. C. and C. V. Pasupulet Maydica 27:213-220 (1982)). Among themore useful traits for which Zea diploperennis may serve as a sourceinclude, resistance to maize chlorotic dwarf, maize chlorotic mottle,and maize streak viruses, and maize bushy stunt mycoplasma as well astolerance to maize raydo fino virus. Unfortunately, even though Zeadiploperennis possesses valuable genetic potential for corn improvement,by being limited to conventional breeding techniques, it will requireyears to develop the improved lines.

It is, therefore, highly desirable to discover conditions which willpermit the regeneration of Zea diploperennis from tissue culture therebyallowing the full range of biotechnological techniques to be brought tobear on the breeding process; thereby significantly reducing the timerequired to recover improved breeding lines.

The culture of the diploid and annual teosinte (Zea mexicana) wasundertaken (Cure, W. W. and R. L. Mott Physiol Plant. 42:91-96 (1978))and limited callus-like growth was reported but no shoot regenerationoccurred. It was also reported (Dhaliwal, H. S. and H. Lorz, MaizeGenetics Coop. Newsletter 53:144 (1979)) that scutella cultures ofimmature embryos of F₁ hybrids between teosinte (Z. mexicana) `ElSalado` and the inbred corn line `B-73` regenerated numerous plantlets.Shoot culture was also tested with mature seeds of B-73, El Salado andB-73 x El Salado. B-73 corn tissues had no response while El Salado andhybrid tissues gave similar results as those described for immatureembryo cultures.

It has not heretofor been possible to employ somatic tissue from fieldgrown plants of Zea diploperennis as a source of material for plantregeneration from tissue culture.

The term "plant tissue culture" as used herein is taken in its broadestmeaning to refer to the cultivation, in vitro, of all plant parts,whether a single cell, a tissue or an organ, under aseptic conditions.More restrictive terms relating to plant tissue culture technologyinclude: "callus culture" by which is meant, the culture of cell masseson agar medium and produced from an explant of a seedling or other plantsource; "cell culture" by which is meant, the culture of cells in liquidmedia in vessels which are usually aerated by agitation; "organ culture"by which is meant, the aseptic culture on nutrient media of anthers(microspores), ovaries, roots, shoots, or other plant organs; "meristemculture and morphogenesis" by which is meant, the aseptic culture ofshoot meristems or other explant tissue on nutrient media for thepurpose of growing complete plants, and "protoplast culture" by which ismeant, the aseptic isolation and culture of plant protoplasts fromcultured cells or plant tissue.

BRIEF DESCRIPTION OF THE INVENTION

This invention provides a method for the controlled regeneration ofhybrids of Zea diploperennis comprising:

forming callus from explant tissue on a first callus promoting medium

subculturing said callus

inducing plantlet regeneration by transferring said callus to a secondregeneration promoting medium

inducing shoot and root formation by transferring said plantlets to athird root promoting medium.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 - This figure illustrates the combination of the tissue cultureand breeding strategies for corn improvement.

FIG. 2 - This figure illustrates a protocol for gene transfer in corn.

DETAILED DESCRIPTION OF THE INVENTION

On their face, the principles underlying plant tissue culture are quitesimple. Initially, it is necessary to isolate a plant part from theintact plant and disrupt its organ, inter-tissue, or inter-cellularrelationships. Subsequently, it is necessary to provide the isolatedmaterial with the appropriate environment in which to express itsintrinsic or induced developmental potential. Finally, the steps must becarried out aseptically. Although the principles may be simply stated,as a matter of practice, the successful culture of plant tissue and itsregeneration into a mature plant is extremely complex.

The regeneration may be envisioned to comprise three stages. The firststage occurs following the transfer of an explant onto a culture medium.This stage is characterized by a proliferation of the explant or callus.The second stage is characterized by a rapid increase in organ growth.This stage may require a transfer to a second medium with or without achange in growth regulator concentration. The final stage occurs whenthe plants are removed from in vitro culture and requires theestablishment of the autotrophic state.

A number of experimental parameters must be addressed during theregeneration protocol. For example, for a particular species the sourceof the explant may be important for the success of the subsequentregeneration. The size and the shape of the explant may also becritical. Another element to be considered is the method of providingaseptic explant material for purpose of callus formation. This involvessterilization of the explant tissue prior to inoculation ontopropagation medium. Even this apparently routine process is subject to awide variety of critical experimental parameters.

It is an object of this invention to provide a method for theregeneration of somatic cells of Zea diploperennis hybrids or hybridsderivatives from tissue culture. Zea diploperennis hybrid derivitives asused herein refers to the progeny of from 1 to 5 backcrosses and/orselfings of a Zea diploperennis x commercial corn variety (Zea mays) F₁plant. It is a further object to use Zea diploperennis as a geneticbridge to introduce not only disease resistance and other agronomicallyuseful traits such as high tillering capacity into a wide variety ofcommercial lines of corn but to transfer plant regeneration capacity tosaid commercial lines as well. Other embodiments of this inventioninclude: The use of F₁ or backcross (BC) plants of Zea mays x Zdiploperennis as donor tissue to generate and recover somaclonalvariants from regenerated somatic cells; and the use of plasmid-like DNAin the mitochondria of Zea diploperennis as a vector for recombinantDNA-based technology.

Apical stem sections of field grown teosinte plants (Zea diploperennis)were collected as donor tissue and external leaves and leaf sheaths wereremoved. Stem segments of 5-8 cm were cut, rinsed for 3 min. with 1%laboratory detergent and washed twice with tap water. These small stemsegments were then transferred to a fungicide solution (Captan 0.5 g/1)for 30 min. with agitation. After three rinsings with sterile water, thestem segments were sterilized in a solution of 2.5% sodium hypochloritefor 30 min. and then dissected. The apical and lateral sub-meristems(3-4 leaf primordia; ca. 1-3 mm long) were aseptically excised using astereoscope. The meristem explants were placed on a basal salts medium(See Table I) containing sucrose (2.5 or 10%), pyridoxine (5 μM),thiamine HCl (1 μM), nictotinic acid (1 μM), inositol (50 μM), agar(0.7%) and various concentrations of 2,4-D between 2.5 and 20 μM. The pHwas adjusted to 5.8 before autoclaving. A filter sterilized solution ofL-asparagine (1 μM) was added to some replicates of each 2,4-Dtreatment. Thirty replicates for each treatment were incubated in thedark at 27°± 1° C. and transferred to fresh medium every 21-18 days.Plant regeneration was obtained on secondary MS medium devoid of 2,4-Dgrown under 2:1 fluorescent/incandescent light (8-10 W/m²), 16 hphotoperiod.

Although several additional basal salts media are known including butnot limited to, B5 medium, White's medium and Schenk-Hilderbrandt medium(See, e.g., Gamborg, O. L. et al., (1976), In Vitro 12: 473-478); thepreferred medium is that described by Murashige, T. and Skoog, F.,(1962), Physiol. Plant. 15: 473-497 (MS medium).

                  TABLE 1    ______________________________________    COMPOSITION OF MS SALT MEDIUM    (MURASHIGE, T., and, F. SKOOG, PHYSIOL.    PLANT. 15: 473-497 (1962))    ______________________________________    MACRO-    NUTRIENTS        mM      mg/l    ______________________________________    NH.sub.4 NO.sub.3                     20.6    1650    KNO.sub.3        18.8    1900    CaCl.sub.2.2H.sub.2 O                     3.0     440    MgSO.sub.4.7H.sub.2 O                     1.5     370    KH.sub.2 PO.sub.4                     1.25    170    ______________________________________    MICRO-    NUTRIENTS        μM   mg/l    ______________________________________    KI               5       0.83    NH.sub.3 BO.sub.3                     100     6.3    MnSO.sub.4.4H.sub.2 O                     100     23.3    MnSO.sub.4.H.sub.2 O                     --      --    ZnSO.sub.4.7H.sub.2 O                     30      8.6    Na.sub.2 Mo.sub.4.2H.sub.2 O                     1.0     0.25    MoO.sub.3        --      --    CuSO.sub.4.5H.sub.2 O                     0.1     0.025    CoSO.sub.4.6H.sub.2 O                     0.1     0.025    Fe.sub.2 (SO.sub.4).sub.3                     --      --    Na.sub.2 EDTA    100     37.3    FeSO.sub.4.7H.sub.2 O                     100     27.8    ______________________________________

The proliferation of callus tissue from submeristem explants of diploidperennial teosinte was observed in 25-30 day old cultures in mediasupplemented with 2.5 to 40 μM 2, 4-D. As this species has multipletillers, it is possible by removing explants from one stem to obtainsomatic tissue regeneration without destroying the donor plant. Immatureinfluorescence is often used as a somatic explant to establish cellcultures of Gramineae. However, stem tissue, available throughout thegrowth cycle, is a more suitable donor tissue than immatureinfluorescence which is only available during the flowering period.

Suitable callus proliferation and survival was only observed in culturesgrown on the 2,4-D containing media at 2.5 to 10 μM concentrations. Nosignificant differences were found in the quality of callus formationfrom cultures growing in this range of 2,4-D concentrations. Callusformation in primary medium was very limited at 20-40 μM 2, 4-D orfailed to proliferate 75-200 μM 2, 4-D. For all treatments, low levelsof sucrose (2%) provided better callus initiation and proliferation thanhigh levels of sucrose (20%). L-asparagine had no influence on callusgrowth.

During first to second subcultures in the primary medium, the tissue hasa white-creamish color and a semi-friable consistency. Callus cultureswith a watery appearance did not grow further under a regime of periodicsubcultures. Semi-friable callus ca. 1-2 cm in diameter was observedafter 50-60 days in culture (second subculture). Callus tissues becamemore friable after 3-4 subcultures on primary medium and some culturescould be identified that contained areas of white tissue that wereslightly different from the rest of the callus mass.

Plant regeneration was induced from small callus pieces placed onsecondary medium (MS Salts, minus 2,4-D) in the presence of light. Onthis medium, numerous adventitious buds were observed in 30% ofcultivated flasks after a few days. The growth and development of theseadventitious buds led to the development of multiple shoots (5-6plants/culture) with the light-green leaves. Curled and wrinkled whiteleaves were frequently observed. These white tissues and leaves werevery similar to the ones observed for Zea mays cultures initiated fromimmature embryos in medium containing 2,4-D (Green, C. E. and R. L.Phillips, Crop Sci. 15: 417-21 (1975)). Green leafy structures wereobserved before he development of shoots. These leaf structures havebeen described as scutella of precociously germinating embryos in tissuecultures of wheat and pearl millet.

Plantlets with first leaves 1-2 cm long were transferred to a tertiarymedium (half-strength MS, with 2% sucrose). A few days later, severalshoots were observed including a few that already initiated roots.Isolated shoots were rooted in the same tertiary medium. In a"potting-up" procedure, the regenerated plants were removed from agarjars, washed thoroughly in tap water, and immediately transferred to aPromix soil mixture in 4 gallon pots. Under greenhouse conditions, thetissue-culture-derived plants grew very fast reaching ca. 80 cm heightafter 50 days. At this time, 11-16 multiple tillers were observed amongthe regenerated greenhouse plants.

The development of high frequency plant regeneration from culturedsomatic tissues of Zea diploperennis as described above offers newpossibilities for plant regeneration from protoplast fusion products ofwild species and commercial lines of corn as well as F₁ and BC₁ progenyof Zea diploperennis and commercial lines of corn. These possibilitiesare predicated on the observation that plant regeneration appears tobehave as a dominant trait in interspecies hybrids with teosinte, thusconstruction of such hybrids would facilitate the selective transfer ofgenes governing regeneration into commercial cultivated corn varieties.Introduction of F₁ hybrids of barley into culture has already been shownto result in chromosome loss in regenerated plants (Orton, T. J.,Theoret. Appl. Genet. 56:101-112 (1980)).

The F₁ or BC₁ plants of corn x Z. diploperennis are also useful as donortissue to recover novel somaclonal variation. Somaclonal variation hasbeen reported among plants regenerated from immature embryos of corn andboth single gene mutations and cytoplasmic DNA changes were observed(Edallo, S. et al. Maydica 26:39-56 (1981) and Umbeck, P. F., and B. G.Gengenbach, Crop Science 23:584-588)). In addition, this inventioncontemplates the recovery of new types of recombinants in plantsregenerated from anthers of F₁ hybrids or BC₁ plants.

Employing the regeneration protocol described above, conventionalbleeding may be coupled with somaclonal variation to establish newbreeding lines. The culture of somatic tissues of segregating sexualhybrids and backcrosses of corn and teosinte are particularly useful inthis regard. As outlined in this scheme (FIG. 1) the most suitable BClor mazoid or teosoid backcrossed plants are identified in the field andselected plants are used as donor tissue for somaclonal variation usingmature tissues. Somaclonal variants are selected for processingcharacters and agronomically important traits such as diseaseresistance.

The following steps comprise the selection strategy for corn improvementand are illustrated in FIG. 1.

(1) Identify superior corn and Z. diploperennis germplasm. Cross parentlines to produce an F₁ hybrid.

(2) Backcross hybrid to maize.

(3) Self-fertilize backcrosses for one to five generations and selectfor mazoid and teosoid type plants.

(4) Introduce stem segments or immature embryos of donor tissue intocell culture.

(5) Regenerate plants and self fertilize to identify somaclonalvariants.

(6) Select desirable somaclones from replicated field plots.

To illustrate this aspect of the invention further, a cross of Zea maysc.v. maya x Zea diploperennis was made employing Z. diploperennis as themale parent. The F₁ was backcrossed to the Zea mays parent and theprogeny selfed for two further generations. The resultant progeny whereclassified as mazoid or teosoid type depending on whether the overallphenotype was more like maize or more like teosinte. Some of theresulting teosoid progeny were further crossed to a Zea mays sugaryvariant and the resulting hybrid was denominated a teosoid hybrid(sugary type).

The Zea mays parental type, the teosoid hybrids resulting from the twoselfing of the F₁ backcross hereinafter referred to as the teosoidhybrid (normal type) and the teosoid hybrid (sugary type) were eachselfed and immature embryos recovered as donor tissue and introducedinto tissue culture as described above for Zea diploperennis Table 2shows the regeneration frequencies for various Zea diploperennis hybridsas compare to the Zea mays control.

Although immature embryos were employed as donor tissue, as demonstratedabove somatic nonembryo tissues, such as apical stem sections, are alsouseful as sources of donor tissue.

    ______________________________________    REGENERATION FREQUENCIES OF MAIZE AND    TEOSINTE/MAIZE HYBRID TISSUES                           Regenerating           No.    Total    callus     Total No.    Genotype explants no. callus                               No.   %    green shoots    ______________________________________    MAIZE    40        46       0     0    0    (dent type)    TEOSOID  187      368      93    25   129    HYBRID    (normal type)    TEOSOID  46       133      65    49   105    HYBRID    (sugary type)    ______________________________________

In a further embodiment, the regeneration protocol supra is employedwith recombinant DNA techniques to achieve genome modification in corn.

Zea diploperennis is particularly attractive for such recombinant DNAstudies due to the recent report of plasmid-like mitochondrial DNA inthis species (Timothy, D. H. et al. Maydica 28:139-49 (1983)). A schemedemonstrating the gene transfer utilizing the plasmid-like DNA isdepicted in FIG. 2. Plasmid-like DNAs are isolated from mitochondria,genetically modified, and then reintroduced into corn organelles.

The following steps comprise the transfer strategy and are illustratedin FIG. 2.

(1) Plasmid-like DNA occurring in Z. diploperennis mitochondria isisolated.

(2) Donor DNA is combined with plasmid-like DNA to produce new hybridDNA.

(3) The hybrid DNA is reintroduced into isolated corn mitochondria.

(4) The isolated, modified mitrochondria are reintroduced into corn ofZ. diploperennis protoplasts.

(5) The modified protoplasts are regenerated into new plants containingmodified DNA.

The plasmid-like DNA may be isolated according to the procedure foTimothy et al. supra, and the hybrid DNA molecules are produced bymethods well-known to workers in the art. See for example MolecularCloning: A Laboratory Manual, T. Maniatis et al., Cold Spring HarborLaboratory, 1982, the contents of which we incorporated herein byreference. Briefly the procedure comprises subjecting both the donor DNAand the plasmid-like DNA to the action of restriction endonucleases,mixing the DNAs to permit the association of the donor DNA with theplasmid-like DNA and ligating the recombinant molecules by treatmentwith DNA ligase.

Once the hybrid DNA is reintroduced into mitochondria and the modifiedmitochondria are subsequently reintroduced to Z. diploperennisprotoplasts; the modified protoplasts are regenerated according to themethod described herein.

What is claimed is:
 1. A method for the controlled regeneration of Zeadiploperennis interspecific hybrids or hybrid derivitivescomprising:forming callus from explant tissue obtained from said hybridsor hybrid derivatives on a first callus promoting medium supplementalwith 2,4-D; subculturing said callus; inducing plantlet regeneration bytransferring said callus to a second regeneration promoting mediumcharacterized by the absence of 2,4 -D; and inducing shoot and rootformation by transferring said plantlets to a third root promotingmedium.
 2. The method of claim 1 which includes the further step ofrecovering the regenerated plantlet from the rooting medium andpotting-up same to provide corn plants.
 3. The method according to claim1 wherein said first callus promoting medium comprises a Murashige-Skoogsalts medium supplanted with sucrose, pyridoxine, thiamine, nicotinicacid, inositol, 2,4-dichlorophenoxy acetic acid (2,4-D), and agar. 4.The method according to claim 3 wherein the 2,4-D is present in aconcentration for about 1 to about 40 μM.
 5. The method according toclaim 4 wherein the 2,4-D is present in a concentration from about 1 toabout 10 μM.
 6. The method according to claim 3 wherein the sucrose ispresent in an amount from about 2% to about 10%.
 7. The method accordingto claim 6 wherein the sucrose is present in an amount of about 2%. 8.The method of claim 1 wherein the callus forming step is conducted inthe dark.
 9. The method of claim 1 wherein the subculturing stepcomprises 1 to 5 subculturing periods on callus promoting medium. 10.The method of claim 9 where a single subculturing period comprisingculturing the callus on said callus promoting medium for a period offrom about 25 to about 30 days.
 11. The method of claim 1 wherein theregeneration promoting medium comprises a Murashige-Skoog salts mediumsupplement with sucrose, pyridoxine thiamine, nicotinic acid, inositoland agar.
 12. The method of claim 11 wherein the sucrose is present inan amount from about 2% to about 10%.
 13. The method of claim 12 whereinthe sucrose is present in an amount of about 2%.
 14. The method of claim1 wherein the plantlet regeneration step is conducted partially in thelight.
 15. The method of claim 14 wherein the regeneration step ischaracterized by a 16 hour photoperiod of 2:1 fluorescent/incandescentlight.
 16. The method of claim 1 wherein said root promoting mediumcomprises one-half strength MS salts medium supplemented with about 2%sucrose.